Recent advances in biotechnology have permitted the development of ELISA tests for various infectious agents. Such testing has become increasingly important, especially for blood screening purposes, to maintain the integrity of hospital blood banks. The introduction of human error, the limited speed of manual processing techniques, and equipment limitations have prevented ELISA tests from achieving their full potential of reliability. Furthermore, preparation and performance of the assay may be tedious when a large number of patient samples are to be tested.
Typically, ELISA tests rely on the use of Microtiter.RTM. plates which have reaction wells coated with a first reactant. Patient sample, in the form of serum or plasma suspected of containing an analyte (i.e., an antibody or antigen) which is capable of specifically binding with the first reactant, is added to the wells. After an incubation period, patient sample and any unbound analyte are removed and the reaction wells carefully washed. A reporter/second reactant conjugate is then added to the reaction wells and incubated. At the end of this second incubation period, unbound conjugate is removed, the wells washed again, and a chromogenic substrate added and allowed to incubate for a third incubation period. A color will then develop in proportion to the amount of analyte which has bound to the first reactant. At the end of the third incubation period, the reactions in each reaction well are stopped by the addition of an acid solution. The optical density of the resulting fluid indicates the quantity of bound analyte, which is indicative of the quantity of the infectious agent or the antibody thereto in the samples. Positive and negative controls are included in the assay to determine a cutoff absorbance, which indicates whether the sample is positive or negative.
The chemical reactions are time-, temperature- and concentration-dependent. Manual methods of conducting ELISA tests invariably result in different processing times for different samples. For example, in a Microtiter.RTM. plate containing 96 reaction wells, 96 patient samples must be prepared. In one test prepared for the detection of acquired immune deficiency syndrome (AIDS) antibodies, patient samples must first be diluted in two steps with a diluent (1:400) before the resulting dilution may be added to the reaction wells. The technician must also accurately identify which patient sample is being placed in which reaction well and usually records this information on a grid which identifies the coordinates of the reaction wells. Preparation of the patient samples, transfer of the samples (or diluted samples), and sample identification can take up to an hour or more for a single assay. Therefore, the reaction between the first reactant and analyte in the first loaded reaction well may have started substantially before the reaction in the last loaded reaction well, resulting in what is known as "front-to-back error."
Similar variations in reactions times may occur, especially if the reaction wells are washed manually and/or loaded with reporter/second reactant conjugate, chromogenic substrate, and stop solution manually. Where automated plate washers are used, it has been found that presently available washers do not completely empty the reaction wells of fluid, requiring the reaction wells to be blotted by the technician.
A further problem in the manual preparation of Microtiter.RTM. plates is cross-contamination between patient samples. Typically, technicians use pipettes for drawing patient sample from test tubes (and for dilution of those samples) which have disposable tips. If patient sample is inadvertently drawn too far into the pipette, disposal of the pipette tip does not prevent contamination of the next sample. It is often difficult for a technician to detect this error or to later identify an anomalous test result as having been caused by this procedural error.
Temperature variations during the incubation periods among the wells in one plate also provide substantial variations in reaction rates in the reaction wells and, therefore, in the optical density of the fluid contained therein. Typically, the Microtiter.RTM. plates are incubated in what are essentially small ovens. These incubators rely primarily on convection to distribute heat evenly among the reaction wells. It is well known that a significant "edge effect" occurs in incubators. This edge effect is the result of a temperature gradient between the center and edges of the plate which is due to the inability of convection currents to evenly heat the plate.
The most serious problem in achieving test reliability and repeatability has been found to be sample misidentification. This error primarily occurs due to transcription errors and sample transfer errors. In the first case, it is known that technicians sometimes incorrectly record the location of a patient sample in a test tube rack. In the second case, technicians have been known to transfer patient samples from a sample test tube to the wrong reaction well in the Microtiter.RTM. plate. Although transcription procedures and handling techniques have been developed to avoid such errors, they are still known to occur. It is possible that the tedious nature of preparing and transferring samples may lead technicians to devote less than their full attention to the task at hand. Once a transcription or sample transfer error has occurred, it is often impossible for the technician to retrace his or her steps to rectify the mistake. Often, the fact that an error has occurred may not be recognized until the assay has been completed and positive results have been impossible to duplicate in a subsequent verification test. In this case, the entire assay must be performed again.
In view of the above, a need exists for a method and an apparatus which substantially reduce the possibility of human error, increase the accuracy, speed and reliability in tests of this type, and which overcome the performance limitations of equipment presently available.